SUMMARY - Core 2 The Extracellular Vesicle Purification and Analysis (EVPA) Core will uniquely serve the three individual projects within the Program Project Grant. Overall, the goal of the Core is centralize the methods, expertise, equipment, and skilled labor to perform extracellular vesicle (EV) purification, analysis, sizing, counting, and quality control. The Core will isolate EVs by differential centrifugation and density-gradient ultracentrifugation using Optiprep/ iodixanol layering. EVs will be counted and sized using nanoparticle tracking analysis. Moreover, this Core will perform Fluorescent Activated Vesicle Sorting (FAVS) which is based on fluorophore-conjugated antibodies to label EV surface markers and genetically encoded fluorescent markers to label internal proteins. This technique allows for the gating (counting) and sorting of EVs which are naturally below the diffraction limit of light based on size. EV preparations will further be sub-fractionated based on specialized density gradients to trace EV protein markers and RNA contents and isolate exomeres. For Project 1, this Core will purify EVs for all 3 Aims, as well as provide EV subfractionation and FAVS EV analysis and sorting for Aim 1C. For Project 2, this Core will purify EVs for all 3 Aims (1-3). For Project 3, this Core will utilize FAVS EV analysis and sorting and EV and exomere purification to achieve the goals of all 3 Aims (1-3). The EVPA Core will also engage in technology development to further develop the FAVS method to analyze single EVs based on fluorescent RNAs and RBPs, and to use fixed, permeabilized EVs for analysis. In summary, the goals of the Core are to provide uniform purification and analysis practices for all 3 projects and uniquely skilled technicians and equipment in a centralized Core. As such, this structure will facilitate extensive but timely characterization and delivery of high quality materials to investigators and drive the cutting edge in this area.